Models of prion disease

ABSTRACT

The present invention provides a novel PrP protein, and nucleic acids encoding this protein, where the PrP protein is characterized in vivo by 1) incomplete glycosylation relative to glycosylation of wild-type PrP C  and 2) proper cellular localization, i.e. an ability to be transported to the cell surface. This novel, under-glycosylated PrP, unlike its normal cellular counterpart, can easily be converted into a protease-resistant isoform by incubation with infectious prions. The invention further provides systems for the study of prion disorders and methods of using these systems, e.g. the study of the mechanical processes in progression of prion-mediated disease or the identification of new therapeutic agents for treatment of prion-mediated disorders. In such systems, protease-resistant under-glycosylated PrP is generated de novo and can be detected by standard immunoblot techniques.

GOVERNMENT RIGHTS

[0001] The United States Government may have certain rights in thisapplication pursuant to Grant No. AG10770 awarded by the NationalInstitutes of Health.

FIELD OF THE INVENTION

[0002] The present invention relates generally to systems for studyingneurodegenerative disorders, and in particular to systems for the studyof prion-associated disease.

BACKGROUND OF THE INVENTION

[0003] Prions are infectious pathogens that cause central nervous systemspongiform encephalopathies in humans and animals. Prions are distinctfrom bacteria, viruses and viroids. The predominant hypothesis atpresent is that no nucleic acid component is necessary for infectivityof prion protein. Further, a prion which infects one species of animal(e.g., a human) will not readily infect another (e.g., a mouse).

[0004] A major step in the study of prions and the diseases that theycause was the discovery and purification of a protein designated prionprotein (“PrP”) [Bolton et al., Science 218:1309-11 (1982); Prusiner etal., Biochemistry 21:6942-50 (1982); McKinley et al., Cell 35:57-62(1983)]. Complete prion protein-encoding genes have since been cloned,sequenced and expressed in transgenic animals. PrP^(C) is encoded by asingle-copy host gene [Basler et al., Cell 46:417-28 (1986)] and isnormally found at the outer surface of neurons. A leading hypothesis isthat prion diseases result from conversion of PrP^(C) into a modifiedform called PrP^(Sc).

[0005] It appears that PrP^(Sc) is necessary for both the transmissionand pathogenesis of the transmissible neurodegenerative diseases ofanimals and humans. See Prusiner, S. B., “Molecular biology of priondisease,” Science 252:1515-1522 (1991). The most common prion diseasesof animals are scrapie of sheep and goats, and bovine spongiformencephalopathy (BSE) of cattle [Wilesmith, J. and Wells, Microbiol.Immunol. 172:21-38 (1991)]. Four prion diseases of humans have beenidentified: (1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3)Gerstmann-Strassler-Scheinker Disease (GSS), and (4) fatal familialinsomnia (FFI) [Gajdusek, D. C., Science 197:943-960 (1977); Medori etal., N. Engl. J. Med. 326:444-449 (1992)]. The presentation of humanprion diseases as sporadic, genetic and infectious illnesses initiallyposed a conundrum which has been explained by the cellular geneticorigin of PrP.

[0006] Most CJD cases are sporadic, but about 10-15% are inherited asautosomal dominant disorders that are caused by mutations in the humanPrP gene [Hsiao et al., Neurology 40:1820-1827 (1990); Goldfarb et al.,Science 258:806-808 (1992); Kitamoto et al., Proc. R. Soc. Lond.343:391-398. latrogenic CJD has been caused by human growth hormonederived from cadaveric pituitaries as well as dura mater grafts [Brownet al., Lancet 340:24-27 (1992)]. Kuru, which for many decadesdevastated the Fore and neighboring tribes of the New Guinea highlands,is believed to have been spread by infection during ritualisticcannibalism [Alpers, M. P., Slow Transmissible Diseases of the NervousSystem, Vol. 1, S. B. Prusiner and W. J. Hadlow, eds. (New York:Academic Press), pp. 66-90 (1979)].

[0007] The initial transmission of CJD to experimental primates has arich history beginning with William Hadlow's recognition of thesimilarity between kuru and scrapie. In 1959, Hadlow suggested thatextracts prepared from patients who died of kuru be inoculated intononhuman primates and that the animals be observed for disease that waspredicted to occur after a prolonged incubation period [Hadlow, W. J.,Lancet 2:289-290 (1959)]. Seven years later, Gajdusek, Gibbs and Alpersdemonstrated the transmissibility of kuru to chimpanzees afterincubation periods ranging form 18 to 21 months [Gajdusek et al., Nature209:794-796 (1966)]. The similarity of the neuropathology of kuru withthat of CJD [Klatzo et al., Lab Invest. 8:799-847 (1959)] promptedsimilar experiments with chimpanzees and transmissions of disease werereported in 1968 [Gibbs, Jr. et al., Science 161:388-389 (1968)]. Overthe last 25 years, about 300 cases of CJD, kuru and GSS have beentransmitted to a variety of apes and monkeys.

[0008] The expense, scarcity and often perceived inhumanity of suchanimal experiments have restricted this work and thus limited theaccumulation of knowledge. While the most reliable transmission data hasbeen said to emanate from studies using nonhuman primates, some cases ofhuman prion disease have been transmitted to rodents but apparently withless regularity [Gibbs, Jr. et al., Slow Transmissible Diseases of theNervous System, Vol. 2, S. B. Prusiner and W. J. Hadlow, eds. (New York:Academic Press), pp. 87-110 (1979); Tateishi et al., Prion Diseases ofHumans and Animals, Prusiner et al., eds. (London: Ellis Horwood), pp.129-134 (1992)].

[0009] The importance of understanding the conversion of PrP^(C) intoPrP^(Sc) has been heightened by the possibility that bovine prions havebeen transmitted to humans who developed variant Creutzfeldt-Jakobdisease (vCJD), G. Chazot, et al., Lancet 347:1181 (1996); R. G. Will,et al., Lancet 347:921-925 (1996). Earlier studies had shown that theN-terminus of PrP^(Sc) could be truncated without loss of scrapieinfectivity, S. B. Prusiner, et al., Biochemistry 21:6942-6950 (1982);S. B. Prusiner, et al., Cell 38:127-134 (1984) and correspondingly, thetruncation of the N-terminus of PrP^(Sc) still allowed its conversioninto PrP^(Sc) (M. Rogers, et al., Proc. Natl. Acad. Sci. USA90:3182-3186 (1993)).

[0010] Recent studies have advanced our ability to visualize thestructural transition of PrP^(c) to PrP^(Sc) at a molecular level. Forexample, the N-terminal portion of PrP^(C) is relatively unstructuredand flexible, but assists in stabilizing structural elements within theC-terminal portion. D. G. Donne et al., Proc. Natl. Acad. Sci. USA94:13452-13457 (1997). Furthermore, immunological studies havedemonstrated that N-terminal epitopes are cryptic in PrP^(Sc),supporting the idea that this region undergoes profound conformationalchange during prion propagation. Peretz et al., J. Mol. Biol.273:614-622 (1997).

[0011] Despite these advances, our understanding of the structuralbiology of the pathogenic conversion process remains incomplete in manyways. For example, it is unknown exactly which structural regions ofPrP^(C) are necessary or sufficient for conformational change to occur.It is also unknown which regions of PrP^(Sc) are necessary or sufficientfor infectivity. Evidence indicates that prion strain phenomena andspecies barriers are a result of different PrP conformations, but theprecise structural determinants of these conformations have not yet beenprecisely identified. Telling et al. Science 274:2079-2082 (1996);Billeter, et al., Acad. Sci. USA 94:7281-7285 (1997).

[0012] Recent studies have identified four residues of mouse PrP (MoPrP)that appear to interact with protein X, a putative factor postulated tofacilitate the conformational change from PrP^(C) to PrP^(Sc). Telling,et. al. Cell 83:79-90 (1995). All four amino acids come together to formthe putative protein X binding site in the tertiary structure ofrecombinant PrP 90-231 and PrP 29-231. D. G. Donne et al., Proc. Natl.Acad. Sci. USA 94:13452-13457 (1997); T. L. James et al., Proc. Natl.Acad. Sci. USA 94:10086-10091 (1997). However, despite several reportsof proteins which bind PrP^(C), the identity of protein X remainselusive. Finally, although the 5 structures of refolded, recombinant PrPmolecules may resemble PrP^(C), a structural solution for PrP^(Sc)remains lacking.

[0013] One method of studying prion disease and the physiologicalchanges inherent in the disease is to alter the physical structure ofthe PrP^(C) protein expressed in infected cells to examine the effect onprogression of prion-mediated disorders. In particular, theglycosylation sites of PrP^(C) were initially examined to determinetheir role in conversion of PrP^(C) to PrP^(Sc). PrP^(C) mutants withThr to Ala substitutions in two NXT consensus glycosylation sites (182and 198) exhibited increased sporadic conversion to a proteinaseK-resistant form following transfection of the mutant constructs incells, suggesting that the molecule possessed decreased conformationalstability and was therefore more likely to undergo the conversion toPrP^(Sc) Taraboulos et al., PNAS, 87:8262-8266 (1990). The PrP^(C)molecule also exhibited aberrant intracellular localization, however,and it was hypothesized that the conversion to PrP^(Sc) could also bedue to the location of the precursor mutant PrP^(C) molecule. The latterhypothesis was strengthened by the expression of a PrP^(C) with a T183Aglycosylation mutation in transgenic mice, which resulted in an altereddistribution of PrP^(C) and an incubation period of>500 20 followinginfection with mouse prions. DeArmond et al., Neuron, 19:1337-1348(1997). The aberrant trafficking of this mutant form of PrP^(C) resultedin accumulation of the PrP^(C) in the cell body, and a complete absenceof PrP^(C) in the dendritic trees. Since PrP^(Sc) formation is thoughtto occur on the cell surface, the aberrant trafficking presumablyprevented PrP^(Sc) formation and accumulation in these transgenicanimals, resulting in the increased incubation period.

[0014] Given the time and cost limitations of presently availablesystems, there is a need in the art for a method of studying thepathogenic conversion process of prion disease in more efficient,time-effective systems. There is thus a need in the art for systems tostudy prion disease using a time-efficient model for prion infection andprogression of prion-mediated disorders.

SUMMARY OF THE INVENTION

[0015] The present invention provides a novel PrP protein, and nucleicacids encoding this protein, where the PrP protein is characterized invivo by 1) incomplete glycosylation relative to glycosylation ofwild-type PrP^(C) and 2) sufficient proper cellular localization, i.e.an ability to be transported to the cell surface in an amount sufficientto allow infectivity. This novel, under-glycosylated PrP, unlike itsnormal cellular counterpart, can be converted into a protease-resistantisoform by incubation with infectious prions. The invention furtherprovides systems, in vitro, cellular and animal, for the study of priondisorders and methods of using these systems, e.g. the study of themechanical processes in progression of prion-mediated disease or theidentification of new therapeutic agents for treatment of prion-mediateddisorders. In such systems, protease-resistant under-glycosylated PrP isgenerated de novo and can be detected by standard immunoblot techniques.

[0016] In one embodiment, the under-glycosylated PrP of the invention isexpressed in cell culture. The conversion of under-glycosylated PrPexpressed in cell culture can be achieved by inoculating dishes of cellswith brain homogenates of prion-diseased animals in as short as 4 days.

[0017] In another embodiment, the under-glycosylated PrP of theinvention is expressed in the brains of transgenic mice. In a preferredembodiment, the expression of the under-glycosylated PrP is controlledby an inducible promoter.

[0018] The present invention also provides non-human transgenic animalsfor the study and diagnosis of prion-mediated pathologies, with thetransgenic animal characterized by a genome artificially altered tocontain an exogenous PrP gene of the invention. In a preferredembodiment, the genome is also altered to contain an inducer sequencewhich effects expression of the exogenous PrP gene. These transgenicanimals are characterized by their ability to develop symptoms of priondisease within 200 days or less after being inoculated with infectiousprion preparations that would normally only infect a genetically diverseanimal. The transgenic animal may additionally have an endogenous genealtered by ablation or modified to express a chimeric form of the gene,and may be either homozygous or heterozygous for these alterations.Preferably, the transgenic animal of the invention is a rat, a hamster,or more preferably a mouse. The genetically diverse animal is preferablya human, a cow, a sheep, a horse, a goat, a deer, a pig, a dog, a cat, aturkey or a chicken.

[0019] The invention also features a method for detecting prions in asample by inoculating a transgenic, non-human mammal, preferably amouse, with material suspected to be contaminated with prions. Theanimal inoculated contains a genome artificially altered to express aunder-glycosylated PrP gene of the invention, and optionally an inducersequence which effects expression of the exogenous PrP gene. Prioninfectivity can be determined by observing the transgenic animal for asymptom of prion disease, such as ataxia. Ataxia may be detected bydirect visual observation of the animals or by means of apressure-sensitive detector positioned under the feet of the mouse,which may distinguish the pattern of a scrapie ill animal relative tothe pattern of an unaffected animal. Alternatively, brain homogenatesfrom the infected animal can be produced and examined for the presenceand/or levels of PrP^(Sc).

[0020] An object of the invention is to provide an ex vivo system forstudying the structural events occurring in conversion.

[0021] Another object of the invention is to provide an ex vivo methodidentifying prions in a sample from a subject suspected of sufferingfrom a prion-mediated disorder.

[0022] Another object of the invention is to provide a method foridentifying cellular factors that mediate PrP activity, interact withPrP and/or facilitate conversion of PrP^(C) to PrP^(Sc).

[0023] Another object is to provide such a transgenic animal which isused as a disease model and/or to assay for the presence of a materialwhich causes disease.

[0024] A feature of the invention is that transgenic cell lines, brainhomogenates and/or animals can be infected with a sample irrespective ofstrain of the infectious prion.

[0025] An advantage of the present invention is that infectivity ofprions in a sample can be determined rapidly.

[0026] These and other objects, advantages, and features of theinvention will become apparent to those persons skilled in the art uponreading the details of the chimeric gene, assay method, and transgenicmouse as more fully described below.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0027] Before the present systems, assays and methods are described, itis to be understood that this invention is not limited to particularmethodologies described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

[0028] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

[0029] The publications discussed herein are provided solely for theirdisclosure prior to the fling date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DEFINITIONS

[0030] The term “Prnp^(0/0) or Prnp-Abl” refers to a transgenic animalwhich has its PrP gene ablated with the “^(0/0)” indicating that bothalleles are ablated whereas o/+ indicates only one is ablated.Specifically, the animal being referred to is generally a transgenicmouse which has its PrP gene ablated i.e., a PrP knockout mouse. In thatthe PrP gene is disrupted no mouse PrP protein is expressed.

[0031] The term “sporadic CJD” abbreviated as “sCJD” refers to the mostcommon manifestation of Creutzfeldt-Jakob Disease (CJD). This diseaseoccurs spontaneously in individuals with a mean age of approximately 60at a rate of I per million individuals across the earth.

[0032] The term “latrogenic CJD” abbreviated as “iCJD” refers to diseaseresulting from accidental infection of people with human prions. Themost noted example of such is the accidental infection of children withhuman prions from contaminated preparations of human growth hormone.

[0033] The term “Familial CJD” refers to a form of CJD which occursrarely in families and is inevitably caused by mutations of the humanPrP gene. The disease results from an autosomal dominant disorder.Family members who inherit the mutations generally succumb to CJD.

[0034] The term “Gerstmann-Strassler-Scheinker Disease” abbreviated as“GSS” refers to a form of inherited human prion disease. The diseaseoccurs from an autosomal dominant disorder. Family members who inheritthe under-glycosylated gene generally succumb to GSS.

[0035] The term “prion” shall mean an infectious particle known to causediseases (spongiform encephalopathies) in animals including cows andhumans. The term “prion” is a contraction of the words “protein” and“infection” and the particles are comprised largely if not exclusivelyof PrP^(Sc) molecules encoded by a PrP gene. Prions are distinct frombacteria, viruses and viroids. Known prions include those which infectanimals to cause scrapie, a transmissible, degenerative disease of thenervous system of sheep and goats as well as bovine spongiformencephalopathies (BSE) or “mad cow” disease and feline spongiformencephalopathies of cats. Four prion diseases known to affect humans are(1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3)Gerstmann-Strassler-Scheinker Disease (GSS), and (4) fatal familialinsomnia (FFI). As used herein prion includes all forms of prionscausing all or any of these diseases or others in any animals used-andin particular in humans, cows and other domesticated farm animals.

[0036] The term “PrP gene” is used herein to describe genetic materialwhich expresses proteins (for example those shown in FIGS. 3-5 of U.S.Pat. No. 5,565,186 issued Oct. 15, 1996) and polymorphisms and mutationssuch as those listed herein under the subheading “Pathogenic Mutationsand Polymorphisms.” The PrP gene can be from any animal including the“host” and “test” animals described herein and any and all polymorphismsand mutations thereof, it being recognized that the terms include othersuch PrP genes that are yet to be discovered.

[0037] The term “PrP gene” refers generally to any gene of any specieswhich encodes any form of a PrP amino acid sequences including any priorprotein, the non-disease form of the protein being referred to asPrP^(C) and the disease form referred to as PrP^(Sc). Some commonlyknown PrP sequences are described in Gabriel et al., Proc. Natl. Acad.Sci. USA 89:9097-9101 (1992) and U.S. Pat. No. 5,565,186 both of whichare incorporated herein by reference to disclose and describe suchsequences.

[0038] The terms “prion preparation”, “preparation” and the like areused interchangeably herein to describe a composition suspected ofcontaining prions obtained from brain tissue of mammals. The mammalsfrom which standardized prion preparations are obtained exhibit clinicalsigns of CNS dysfunction as a result of inoculation with prions and/ordue to developing the disease due to their genetically modified make up,e.g., high copy number of PrP genes.

[0039] The term “artificial PrP gene” is used herein to encompass theterm “chimeric PrP gene” as well as other recombinantly constructedgenes which when included in the genome of a host animal (e.g., a mouse)will render the mammal susceptible to infection from prions whichnaturally only infect a genetically diverse test mammal, e.g., human,bovine or ovine. In general, an artificial gene will include the codonsequence of the PrP gene of the mammal being genetically altered withone or more (but not all, and generally less than 40) codons of thenatural sequence being replaced with a different codon—preferably acorresponding codon of a genetically diverse mammal (such as a human).The genetically altered mammal being used to assay samples for prionswhich only infect the genetically diverse mammal. Examples of artificialgenes are mouse PrP genes encoding the sequence as shown in FIGS. 3 +L,4 and 5 of U.S. Pat. No. 5,565,186, with one or more differentreplacement codons selected from the codons shown in these Figures forhumans, cows and sheep replacing mouse codons at the same position, withthe proviso that not all the mouse codons are replaced with differinghuman, cow or sheep codons. Artificial PrP genes of the invention havemutations that affect glycosylation of the PrP molecule, and: codons ofgenetically diverse animals; codons and/or codon sequences associatedwith genetic prion diseases such as CJD; and codons and sequences notassociated with any native PrP gene but which, when inserted into ananimal, render the animal susceptible to infection with prions whichwould normally only infect a genetically diverse animal. Theintroduction of artificial PrP genes may be stable or transient,particularly for cell transfection assays. For example, the artificialPrP gene may be introduced using a viral vector. In this manner, the PrPgene may be introduced to particular tissues or cell types, e.g. CNStissue.

[0040] The terms “chimeric gene,” “chimeric PrP gene”, and the like areused interchangeably herein to mean an artificially constructed genecontaining the codons of a host animal such as a mouse with one or moreof the codons being replaced with corresponding codons from agenetically diverse test animal such as a human, cow or sheep. Thechimeric PrP genes of the invention all additionally contain mutationsthat decrease the levels of glycosylation of PrP in vivo. In onespecific example the chimeric gene is comprised of the starting andterminating sequence (i.e., − and C-terminal codons) of a PrP gene of amammal of a host species (e.g. a mouse) and also containing a nucleotidesequence of a corresponding portion of a PrP gene of a test mammal of asecond species (e.g. a human). A chimeric gene will, when inserted intothe genome of a mammal of the host species, render the mammalsusceptible to infection with prions which normally infect only mammalsof the second species. The preferred chimeric gene disclosed herein isMHu2M which contains the starting and terminating sequence of a mousePrP gene and a non-terminal sequence region which is replaced with acorresponding human sequence which differs from a mouse PrP gene in amanner such that the protein expressed thereby differs at nine residues.

[0041] The term “genetic material related to prions” is intended tocover any genetic material which affects the ability of an animal tobecome infected with prions. Thus, the term encompasses any “PrP gene”,“artificial PrP gene”, “chimeric PrP gene” or “ablated PrP gene” whichterms are defined herein as well as mutations and modifications of suchwhich effect the ability of an animal to become infected with prions.

[0042] The terms “host animal” and “host mammal” are used to describeanimals which will have their genome genetically and artificiallymanipulated so as to include genetic material which is not naturallypresent within the animal. For example, host animals include mice,hamsters and rats which have their endogenous PrP gene altered by theinsertion of an artificial gene of the present invention.

[0043] The terms “test animal” and “test mammal” are used to describethe animal which is genetically diverse from the host animal in terms ofdifferences between the PrP gene of the host animal and the PrP gene ofthe test animal. The test animal may be any animal for which one wishesto run an assay test to determine whether a given sample contains prionswith which the test animal would generally be susceptible to infection.For example, the test animal may be a human, cow, sheep, pig, goat,deer, horse, cat, dog, turkey or chicken, and one may wish to determinewhether a particular sample includes prions which would normally onlyinfect the test animal. This is done by including PrP gene sequences ofthe test animal into the host animal and inoculating the host animalwith prions which would normally only infect the test animal.

[0044] The terms “genetically diverse animal” and “genetically diversemammal” are used to describe an animal which includes a native PrP codonsequence of the host animal which differs from the genetically diversetest animal by 17 or more codons, preferably 20 or more codons, and mostpreferably 28-40 codons. Thus, a mouse PrP gene is genetically diversewith respect to the PrP gene of a human, cow or sheep, but is notgenetically diverse with respect to the PrP gene of a hamster.

[0045] The terms “ablated PrP gene”, “disrupted PrP gene”, and the likeare used interchangeably herein to mean an endogenous PrP gene which hasbeen altered (e.g., add and/or remove nucleotides) in a manner so as torender the gene inoperative. Examples of non-functional PrP genes andmethods of making such are disclosed in Büeler, H., et al “Normaldevelopment of mice lacking the neuronal cell-surface PrP protein”Nature 356:577-582 (1992) which is incorporated herein by reference.Both alleles of the genes are preferably disrupted.

[0046] The terms “hybrid animal”, “transgenic hybrid animal” and thelike are used interchangeably herein to mean an animal obtained from thecross-breeding of a first animal having an ablated endogenous PrP genewith a second animal which includes a chimeric gene or artificial PrPgene of the invention. For example a hybrid mouse is obtained bycross-breeding a mouse with an ablated mouse PrP gene with a mousecontaining under-glycosylated chimeric PrP genes. The term hybridincludes any offspring of a hybrid including inbred offspring of twohybrids provided the resulting offspring is susceptible to infectionwith prions with normal infect only a genetically diverse species andthe symptoms of the infection are observable in about 350 days or less,preferably 250 or less.

[0047] The terms “susceptible to infection”, “susceptible to infectionby prions”, and the like are used interchangeably herein to describe atransgenic or hybrid test animal of the invention which develops a priondisease if inoculated with prions which would normally only infect agenetically diverse test animal. The terms are used to describe atransgenic or hybrid animal of the invention such as a transgenic mouseTg(MHu2M) which, without the chimeric PrP gene, would not be susceptibleto infection with a human prion (less than 20% chance of infection) butwith the chimeric gene is susceptible to infection with human prions(80% to 100% chance of infection). If an animal is susceptible toinfection with a particular prion that animal, if inoculated with theprion, will show symptoms of prion disease infection in about 350 daysor less, preferably 250 days or less.

[0048] The term “incubation time” shall mean the time from inoculationof an animal with a prion until the time when the animal first developsdetectable symptoms of disease resulting from the infection. A reducedincubation time is one year or less, preferably about 200 days±50 daysor less, more preferably about 50 days±20 days or less.

[0049] Abbreviations used herein include:

[0050] CNS for central nervous system;

[0051] BSE for bovine spongiform encephalopathy;

[0052] CJD for Creutzfeldt-Jakob Disease;

[0053] FFI for fatal familial insomnia;

[0054] GSS for Gerstmann-Strassler-Scheinker Disease;

[0055] Hu for human;

[0056] HuPrP for a human PrP;

[0057] Mo for mouse;

[0058] Bo for bovine;

[0059] MoPrP for a mouse PrP;

[0060] SHa for a Syrian hamster;

[0061] SHaPrP for a Syrian hamster PrP;

[0062] Tg for transgenic;

[0063] Tg(SHaPrP) for a transgenic mouse containing the PrP gene of aSyrian hamster;

[0064] Tg(HuPrP) for transgenic mice containing the complete human PrPgene;

[0065] Tg(ShePrP) for transgenic mice containing the complete sheep PrPgene;

[0066] Tg(BoPrP) for transgenic mice containing the complete cow PrPgene;

[0067] PrP^(Sc) for the scrapie isoform of the PrP;

[0068] MoPrP^(Sc) for the scrapie isoform of the mouse PrP;

[0069] MHu2M for a chimeric mouse/human PrP gene wherein a region of themouse PrP gene is replaced by a corresponding human sequence whichdiffers from mouse PrP at 9 codons;

[0070] MBo2M for a chimeric mouse/bovine PrP gene wherein a region ofthe mouse PrP gene is replaced by a corresponding bovine sequence whichdiffers from mouse PrP at 8 codons.

[0071] Tg(MHu2M) mice are transgenic mice of the invention which includethe chimeric MHu2M gene;

[0072] MHu2MPrP^(Sc) for the scrapie isoform of the chimeric human/mousePrP gene;

[0073] PrP^(CJD) for the CJD isoform of a PrP gene;

[0074] Prnp^(0/0) for ablation of both alleles of an endogenous PrPgene, e.g., the MoPrP gene;

[0075] Tg(BoPrP)/Prnp^(0/0) for a transgenic mouse obtained with abovine PrP gene (BoPrP);

[0076] Tg(MHu2M)/Prnp^(0/0) for a mouse with a chimeric (mouse/human)PrP gene (WHu2M) with both alleles of the endogenous mouse PrP genedisrupted;

[0077] Tg(MBo2M)Prnp^(0/0) for a transgenic mouse with a chimeric(mouse/bovine) PrP gene (MBo2M) with both alleles of the endogenousmouse PrP gene disrupted;

[0078] FVB for a standard inbred strain of mice often used in theproduction of transgenic mice since eggs of FVB mice are relativelylarge and tolerate microinjection of exogenous DNA relatively well.

GENERAL ASPECTS OF THE INVENTION

[0079] The present invention is based on the discovery that expressionof an under-glycosylated PrP^(C) protein in cells renders these cellssusceptible to infection by prions, when the cells are exposed topreparations containing prions, allowing these cells to produce de novoPrP^(Sc). Although previous Thr to Ala substitutions in theglycosylation sites of PrP resulted in conformational instability, theresulting PrP^(C) molecules displayed an aberrant cellular localizationand resulted in a very long prion incubation period when expressed intransgenic mice. Surprisingly, mutations of the present invention, e.g.Asn to Gin mutations in the PrP glycosylation site, result in a PrP^(C)molecule that are conformationally unstable but properly localized atthe cell surface in amounts that allow conversion of PrP^(C) to PrP^(Sc)upon infection. The conversion of the under-glycosylated PrP^(C) israpid, allowing for quick detection of prions in the cells expressingthis variant PrP and allowing inoculation transgenic mice to show signsof infection in 250 days or less.

[0080] Currently, the most sensitive bioassay for prion infection, theintracerebral inoculation, lasts several months even if transgenic miceoverexpressing PrP are used as recipient animals for intracerebralinfection. Diagnosis of transmissibility is made only upon appearance ofcharacteristic symptoms like ataxia, and, when examining the brain,typical neuropathological features of diseased brain tissue such asvacuolation, astrogliosis, and nerve cell loss. In vitro methods ofdiagnosing prion disease in a suspected diseased brain, such asdetection of the presence of protease-resistant PrP on Western blots, islimited by the sensitivity of the immunological assay. The presentinvention provides an ex vivo method of detection, combining thesensitivity and specificity of an animal bioassay and the rapidity of aWestern blot procedure.

[0081] Cellular systems expressing under-glycosylated PrP^(C) providemethods of transient or permanent overexpression of anunder-glycosylated PrP in a particular cell line, incubation of thesecells with the tissue suspension or body fluid of interest, andexamination of the de novo formation of prions with a conventionalmethod like Western blotting allows detection of prions in a samplewithout the limitations found in currently available bioassays. Therapid replication of prions in this cell line overexpressingunder-glycosylated PrP is used to amplify minute amounts of prionspresent in various tissues.

[0082] In one embodiment, the PrP encodes a double mutation where thetwo asparagines in the glycosylation sites are substituted with anotheramino acid. In a preferred embodiment, the two asparagines aresubstituted to glutamine (N180Q/N196Q). The under-glycosylated PrPovercomes the problems of cell trafficking while still preventingglycosylation. Correct transportation of this under-glycosylated PrPinto the same compartments as its wild-type counterpart has beenexperimentally shown by immunofluorescence and co-transfection studies.

[0083] This rapid bioassay is a major improvement in diagnosing priondiseases, since available bioassays in cell lines were limited toparticular strains of prions and took at least 4 weeks to convertcell-resident PrP. Alternatively, inoculation of available transgenicmice takes 2 to 9 months, depending on the species of prion used(Fischer et al., 1996; Prusiner, 1997; Telling et al., 1994). Thepresent invention of an under-glycosylated PrP overcomes these limits ofprior art in that cell lines can be infected with brain homogenates ofprion-diseased animals irrespective of strains with an unsurpassedrapidity.

PrP Nucleic Acid Compositions

[0084] The term “under-glycosylated PrP” as used within the presentspecification generically designates PrP genes encoding proteins alteredwith respect to their ability to be glycosylated relative to the normalcellular form of PrP. The term encompasses PrP from any species, e.g.homologs from rat, bovine, human, mouse, guinea pig, etc., and theiralternate forms. Used generically, this term encompasses differentisoforms, polymorphisms, mutations and variant sequences, so long as theunder-glycosylated protein's cellular distribution is not affected. Theterm is also intended to mean the open reading frame encoding specificunder-glycosylated polypeptides, introns, and adjacent 5′ and 3′non-coding nucleotide sequences involved in the regulation ofexpression, up to about 1 kb beyond the coding region, but possiblyfurther in either direction. The DNA sequences encoding PrP may be cDNAor genomic DNA or a fragment thereof. The gene may be introduced into anappropriate vector for extrachromosomal maintenance or for integrationinto the host.

[0085] A genomic sequence of interest in the present invention comprisesthe altered nucleic acid present between the initiation codon and thestop codon, as defined in the listed sequences, including all of theintrons that are normally present in a native chromosome. It may furtherinclude the 3′ and 5′ untranslated regions found in the mature mRNA. Itmay further include specific transcriptional and translationalregulatory sequences, such as promoters, enhancers, etc., includingabout 1 kb, but possibly more, of flanking genomic DNA at either the 5′or 3′ end of the transcribed region. The genomic DNA may be isolated asa fragment of 100 kb or smaller; and substantially free of flankingchromosomal sequence.

[0086] The under-glycosylated PrP sequence, including flanking promoterregions and coding regions, may be additionally under-glycosylated invarious ways known in the art to generate targeted changes in promoterstrength, sequence of the encoded protein, etc. The sequence changes maybe substitutions, insertions or deletions. Deletions may include largechanges, such as deletions of a domain or exon. Other modifications ofinterest include epitope tagging, e.g. with the FLAG system, HA, etc.For studies of subcellular localization, fusion proteins with greenfluorescent proteins (GFP) may be used. Such under-glycosylated genesmay be used to study structure-function relationships of prionpolypeptides, or to alter properties of the proteins that affect theirfunction or regulation.

[0087] Techniques for in vitro mutagenesis of cloned genes are known.Examples of protocols for scanning mutations may be found in Gustin etal., Biotechniques 14:22 (1993); Barany, Gene 37:111-23 (1985);Colicelli et al., Mol Gen Genet 199:537-9 (1985); and Prentki et al.,Gene 29:303-13 (1984). Methods for site specific mutagenesis can befound in Sambrook et al., Molecular Cloning: A Laboratory Manual, CSHPress, pp. 15.3-15.108 (1989); Weiner et al., Gene 126:3541 (1993);Sayers et al., Biotechniques 13:592-6 (1992); Jones and Winistorfer,Biotechniques 12:528-30 (1992); Barton et al., Nucleic Acids Res18:7349-55 (1990); Marotti and Tomich, Gene Anal Tech 6:67-70 (1989);and Zhu, Anal Biochem 177:120-4 (1989).

[0088] The mutations present in the PrP of the invention may beintroduced into any genetic background, and the PrP gene itself may haveany number of polymorphisms or specific mutations associated withdisease states. There are a number of mutations and polymorphismsexisting with respect to the PrP gene of different species. A number ofthe mutations and polymorphisms are listed in the “Mutation Table”provided below. It is believed that additional mutations andpolymorphisms exist in all species within the PrP gene. Animals with aPrP gene which is heterozygous at a particular point could be bred withother animals which are heterozygous at that point in order to produceoffspring which include those with a homozygous PrP gene of the typedesired. Substitutions in the PrP transgene may be made with an aminoacid which is biochemically quite different from the amino acid at thatposition which is known to render the animal susceptible to prioninfection. Thus, if a basic and/or polar amino acid is present at thecritical site that site could be replaced with an acidic and/or nonpolaramino acid. With these criteria in mind some trial and error would berequired. Acidic amino acids should be substituted with basic aminoacids and vice versa. Polar amino acids should be substituted withnonpolar amino acids and vice versa. Such mutations may increase thesusceptibility of the transgenic animals for the uses described herein.

[0089] There are a number of known pathogenic mutations in the human PrPgene. Further, there are known polymorphisms in the human, sheep andbovine PrP genes. The following is a list of such mutations andpolymorphisms: MUTATION TABLE Pathogenic human Human Sheep Bovinemutations Polymorphisms Polymorphisms Polymorphisms 2 octarepeat Codon129 Met/Val Codon 171 Arg/Gln insert 4 octarepeat Codon 219 Glu/LysCodon 136 Ala/Val insert 5 octarepeat Codon 154 Arg/His 5 octarepeatinsert insert 6 octarepeat 6 octarepeat insert insert 7 octarepeat 7octarepeat insert insert 8 octarepeat insert 9 octarepeat insert Codon102 Pro-Leu Codon 105 Pro-Leu Codon 117 Ala-Val Codon 145 Stop Codon 178Asp-Asn Codon 180 Val-Ile Codon 198 Phe-Ser Codon 200 Glu-Lys Codon 210Val-Ile Codon 217 Asn-Arg Codon 232 Met-Ala

[0090] In order to provide further meaning to the above chartdemonstrating the mutations and polymorphisms, one can refer to thepublished sequences of PrP genes. For example, a chicken, bovine, sheep,rat and mouse PrP gene are disclosed and published within Gabriel etal., Proc. Natl. Acad. Sci USA 89:9097-9101 (1992). The sequence for theSyrian hamster is published in Basler et al., Cell 46:417-428 (1986).The PrP gene of sheep is published by Goldmann et al., Proc. Natl. Acad.Sci. USA 87:2476-2480 (1990). The PrP gene sequence for bovine ispublished in Goldmann et al., J. Gen. Virol. 72:201-204 (1991). Thesequence for chicken PrP gene is published in Harris et al., Proc. Natl.Acad. Sci. USA 88:7664-7668 (1991). The PrP gene sequence for mink ispublished in Kretzschmar et al., J. Gen. Virol. 73:2757-2761 (1992). Thehuman PrP gene sequence is published in Kretzschmar et al., DNA5:315-324 (1986). The PrP gene sequence for mouse is published in Lochtet al., Proc. Natl. Acad. Sci. USA 83:6372-6376 (1986). The PrP genesequence for sheep is published in Westaway et al., Genes Dev. 8:959-969(1994). These publications are all incorporated herein by reference todisclose and describe the PrP gene and PrP amino acid sequences.

[0091] The under-glycosylated PrP nucleic acids of the invention mayalso encode a known epitope, such as a polyhistidine or c-myc epitopetag, which is inserted into an expressed region of the sequence. Theepitope is inserted such that it is in-frame and thus properlytranslated. The epitope sequences may be added in-frame to various sitesof the full-length protein. Preferably, the exogenous epitope isinserted in-frame carboxy to the end of the translated PrP polypeptide.

[0092] The epitope tag sequences may alter solubility of the encoded PrPas well, and this ability may be position dependent. Thus varying thesite may alter the solubility, conformation, and activity of thefull-length protein.

[0093] Any epitope tag currently known in the art may be used, althoughpreferably the epitope used is comprised of charged residues, and morepreferably is a polyhistidine tag. The epitope tag is preferablycomprised of polar weakly basic residues such as arginine, or lysine andmore preferably histidine. Polar residues of any type would be useful inincreasing solubility.

Cellular Systems Of The Invention

[0094] The present invention provides a cellular system for the studyand diagnosis of prion-mediated diseases, and methods of using suchcellular systems. The cellular systems allow for the study of mechanismsinvolved in prion infection and disease without having to use an animalmodel. Cellular systems of the invention offer an advantage in that theyare less costly than animal systems, offer faster results than animalsystems, and are more humane since they do not require the sacrifice ofanimals to study the cellular mechanisms of the disease progression.

[0095] Knowledge of the conditions under which scrapie infectivity isgenerated de novo is useful in the development of assays to identifycompounds able to inhibit the generation of PrP^(Sc). Compounds able toinhibit the ex vivo conversion of PrP^(C) to PrP^(Sc) could be usefulfor the treatment and prevention of prion-mediated diseases in animaland human subjects at risk. Such cellular systems provide numerousmethods for investigation, including: an ex vivo mechanism to study thephysiological basis and progression of the disease, e.g. molecularinteractions involved in prion infection and the progression ofprion-mediated disease; methods for identifying cellular factors thatinteract with PrP^(C) and/or PrP^(Sc); diagnosis of samples from animalsthat are potentially infected with prions without having to sacrifice ananimal in the process; and assays to examine the efficacy and toxicityof potential therapeutic agents that block prion infection and/or theprogression of a prion-mediated disorder. These and other uses for thecellular model of prion infection will be obvious to one skilled in theart upon reading this disclosure.

[0096] The PrP of the invention may be expressed in any cell type,including prokaryotic cells, such as E. coli, and eukaryotic cells,including cells from S. cerevisae, D. melanogaster, X laevis, aviancells, mammalian expression systems, and the like. Expression of the PrPof the invention finds particular use with mammalian cells ,such as CHOand COS cells, since these cells have mammalian cellular components andare particularly useful for the study and diagnosis of prion-mediateddisorders. Preferably, the cells for use in a cellular system of theinvention are neuronal mammalian cells, and more preferably N2a cells.The cells may be transiently transfected or, more preferably, stablytransfected and maintained in long-term culture. Exemplary methods fortransfection of cells with PrP nucleic acids can be found in Sambrook etal., Molecular Cloning: A Laboratory Manual (3rd ed.) which isincorporation by reference herein to aid one skilled in the art in thesetechniques.

[0097] In one embodiment of the invention, a sample may be assayed forthe presence of infectious prions by exposing a sample to be tested to acellular system expressing the under-glycosylated form of PrP, allowingfor sufficient incubation time, and then assaying the culture for thepresence of prions.

[0098] In another embodiment, the PrP of the invention is transfecteddirectly into the cells of the sample to be assayed, and the level ofprions in these cells determined after an appropriate incubation period.The ability of the PrP of the invention to readily transform into theinfectious form of PrP allows primary cultures to be manipulated andassayed directly following transfection of under-glycosylated PrP intosuch cells.

Detection of Prion Protein in a Sample

[0099] Once a sample is tested in the ex vivo system of the invention,sample may be assayed by a variety of known techniques. The examples andcomparative examples put forth herein utilize a conformation-dependentimmunoassay (CDI) of the type which is disclosed and described withinPCT Publication WO 98/37411 published Feb. 20, 1998. However, othertypes of protein detection assays could be utilized. For example, thesample can be assayed using antibodies of the type disclosed anddescribed within U.S. Pat. No. 5,846,533 issued Dec. 8, 1998, which isincorporated herein by reference in its entirety in order to discloseand describe specific types of assays which might be used on the samplesprepared in accordance with the present invention.

[0100] The method requires beginning with a sample which is divided intotwo portions. The first portion of the sample is contacted with abinding partner. The binding partner has a higher affinity for the firstconformation of the protein than it has for the second pathogenicconformation of the protein. The binding partner is typically anantibody such as labeled 3F4. After allowing the binding partner to bindto a protein in the first conformation the concentration of the bindingpartner/protein complexes formed is determined.

[0101] A second portion of the sample is then treated in a manner whichcauses the binding affinity of the protein in the second conformation tobe enhanced with respect to the binding partner. This treatment caninclude a variety of different methodologies and often involves exposureof the sample to a protease for a sufficient period of time undersufficient conditions so as to cause the protein in the secondpathogenic conformation to become more relaxed and therefor more likelyto bind to the binding partner.

[0102] The treated second portion is then brought into contact with thebinding partner. After binding between the proteins and binding partnersare allowed to occur the concentration of binding partner/proteincomplexes formed in this second treated portion is determined.

[0103] If the sample contained no protein in the second, pathogenicconformation of the protein then the treatment will have little effect.However, the treatment will have some effect on the first conformationof the protein and is likely to increase it's binding affinity to thebinding partner in some small degree. Accordingly, an adjustment must bemade in the second concentration in order to provide an adjustedconcentration which adjustment compensates for the increased affinity ofthe protein of the first conformation for the binding partner resultingfrom the treatment.

[0104] After obtaining the first concentration and the adjustedconcentration the two are compared to each other. If the adjustedconcentration is greater than the first concentration such is anindication of the presence of protein in the second pathogenicconformation in the original sample.

[0105] It is possible to carry out variations on theconfirmation-dependent immunoassay. One variation which is described indetail in published PCT application WO 98/37411 does not require thatthe original sample be broken into first and second portions. Thetreatment process is carried out on a sample and the concentration isdetermined. That concentration is then compared with a known standard(previously obtained on a statistically significant group of samples) inorder to determine if the sample being tested contains prions.

[0106] However, it is pointed out that the present invention is notlimited to the use of such protein assay methodology. Other assays couldbe used and other assays will, no doubt, be developed which couldutilize the samples prepared in accordance with the present invention inorder to obtain accurate results.

Identification of Compounds that Interact with PrP^(C) Or PrP^(Sc)

[0107] In one aspect, the present invention provides novel assays usefulin identifying inhibitors of the formation of a PrP^(Sc)-like complexresulting from PrP peptide binding to PrP^(C). Although ex vivo assaysof the present invention can be configured in a number of ways, in apreferred configuration, a test compound is contacted with cellsexpressing the under-glycosylated PrP^(C). The cell can be examined forprion protein complex in any number of ways, includingimmunoprecipitation. Percent sedimentation, protease resistance, andconformation are determined by methods known in the art, such as thosemethods described below. Formation of a PrP^(Sc)-like complex in thepresence of a test compound is compared to complex formation in theabsence of the test compound (control).

[0108] A compound identified by the assay method of the invention asinhibiting complex formation can be tested in an animal model of aPrP^(Sc)-mediated disease, and its ability to inhibit PrP^(Sc) inductionin vivo or treat a PrP^(Sc)-mediated disease determined. As definedabove, treatment of a PrP^(Sc)-mediated disease includes obtaining atherapeutically detectable and beneficial effect on a patient sufferingfrom a PrP^(Sc)-mediated disease.

[0109] The documented competition of the anti-PrP monoclonal antibody3F4 for the interaction between PrP^(C) and PrP^(Sc) provides analternate strategy for an assay to screen for compounds able to blockprion induction. In one embodiment, PrP^(C) is derivatized, e.g., withStreptavidin, and immobilized to a solid support, for example, thebottom of the wells of a 96-well plate. Candidate compounds are testedfor the ability to displace 3F4 from PrP^(C). Binding is quantitatedthrough standard measures, for example, by determining the amount offree antibody. Variations in this assay include use of variousPrP^(C)-like molecules from recombinant sources.

In vitro Drug Efficacy Evaluation

[0110] The cellular systems of the invention can be used in determinedpotential efficacy of a therapeutic agent directed at prion-mediateddisorders. Compounds can be contacted with cells expressing theunder-glycosylated PrP of the invention, and the effect of this compoundon infectivity, progression of the disease, formation of amyloidplaques, etc., determined. The drug may be determined efficacious if it:(1) increases the rate at which the harmful protein is cleared from thesystem as compared to the rate it is cleared from the system of themouse with no drug; (2) prevents initial prion infection; and/or (3)retards or halts the progression of the prion development. Compoundsthat show promise in an in vitro system can be further studied todetermine their efficacy in vivo.

Transgenic Animals

[0111] The term “transgene” is used herein to describe genetic materialthat has been or is about to be artificially inserted into the genome ofa cell, particularly a mammalian cell for implantation into a livinganimal. The transgene is used to transform a cell, meaning that apermanent or transient genetic change, preferably a permanent geneticchange, is induced in a cell following incorporation of exogenous DNA. Apermanent genetic change is generally achieved by introduction of theDNA into the genome of the cell. Vectors for stable integration includeplasmids, retroviruses and other animal viruses, YACs, and the like. Ofinterest are transgenic mammals, e.g. cows, pigs, goats, horses, etc.,and particularly rodents, e.g. rats, mice, etc.

[0112] Transgenic animals comprise an exogenous nucleic acid sequencepresent as an extrachromosomal element or stably integrated in all or aportion of its cells, especially in germ cells. Unless otherwiseindicated, it will be assumed that a transgenic animal comprises stablechanges to the germine sequence. During the initial construction of theanimal, “chimeras” or “chimeric animals” are generated, in which only asubset of cells have the altered genome. Chimeras are primarily used forbreeding purposes in order to generate the desired transgenic animal.Animals having a heterozygous alteration are generated by breeding ofchimeras. Male and female heterozygotes are typically bred to generatehomozygous animals.

[0113] The exogenous PrP gene is altered to prevent completeglycosylation of the molecule while maintaining proper cellularlocalization of the produced under-glycosylated PrP potein. Themutations to achieve these characteristics may be in any suitablegenetic background. For example, mutations to the glycosylation sitesmay be in a wild type background, in a background with naturallyoccurring polymorphisms, or in a background with genetically manipulatedsequences, e.g. deletions, substitutions or insertions in the coding ornon-coding regions. The introduced under-glycosylated PrP codingsequence is usually operably linked to a promoter, which may beconstitutive or inducible, and other regulatory sequences required forexpression in the host animal. By “operably linked” is meant that a DNAsequence and a regulatory sequence(s) are connected in such a way as topermit gene expression when the appropriate molecules, e.g.transcriptional activator proteins, are bound to the regulatorysequence(s). In some cases the exogenous transgene sequences areultimately deleted from the genome, leaving a net change to the nativesequence.

[0114] In the present invention, transgenic knockouts may additionallyhave a partial or complete loss of function in one or both alleles ofthe endogenous PrP gene. Preferably, the target gene expression isundetectable or insignificant. A knock-out of an endogenous PrP genemeans that function of the PrP protein has been substantially decreasedso that expression is not detectable or only present at insignificantlevels. This may be achieved by a variety of mechanisms, includingintroduction of a disruption of the coding sequence, e.g. insertion ofone or more stop codons, insertion of a DNA fragment, etc., deletion ofcoding sequence, substitution of stop codons for coding sequence, etc.Different approaches may be used to achieve the “knock-out.” See U.S.Pat. Nos. 5,464,764, 5,627,059 and related patents and publications toCapecchi et al. A chromosomal deletion of all or part of the native genemay be induced, including deletions of the non-coding regions,particularly the promoter region, 3′ regulatory sequences, enhancers, ordeletions of gene that activate expression of PrP genes. A functionalknock-out may also be achieved by the introduction of an anti-senseconstruct that blocks expression of the native genes (for example, seeLi and Cohen, Cell 85:319-329 (1996)). “Knock-outs” also includeconditional knock-outs, for example where alteration of the target geneoccurs upon exposure of the animal to a substance that promotes targetgene alteration, introduction of an enzyme that promotes recombinationat the target gene site (e.g. Cre in the Cre-lox system), or othermethod for directing the target gene alteration postnatally.

[0115] DNA constructs for homologous recombination will comprise atleast a portion of the PrP gene with the desired genetic modification,and will include regions of homology to the target locus. DNA constructsfor random integration need not include regions of homology to mediaterecombination. Conveniently, markers for positive and negative selectionare included. Methods for generating cells having targeted genemodifications through homologous recombination are known in the art. Forvarious techniques for transfecting mammalian cells, see Keown et al.,Methods in Enzymology 185:527-537 (1990).

[0116] For embryonic stem (ES) cells, an ES cell line may be employed,or embryonic cells may be obtained freshly from a host, e.g. mouse, rat,guinea pig, etc. Such cells are grown on an appropriatefibroblast-feeder layer or grown in the presence of appropriate growthfactors, such as leukemia inhibiting factor (LIF). When ES cells havebeen transformed, they may be used to produce transgenic animals. SeeU.S. Pat. Nos. 5,387,742, 4,736,866 and 5,565,186 for methods of makingtransgenic animals. After transformation, the cells are plated onto afeeder layer in an appropriate medium. Cells containing the constructmay be detected by employing a selective medium. After sufficient timefor colonies to grow, they are picked and analyzed for the occurrence ofhomologous recombination or integration of the construct. Those coloniesthat are positive may then be used for embryo manipulation andblastocyst injection. Blastocysts are obtained from 4 to 6 week oldsuperovulated females. The ES cells are trypsinized, and the modifiedcells are injected into the blastocoel of the blastocyst. Afterinjection, the blastocysts are returned to each uterine horn ofpseudopregnant females. Females are then allowed to go to term and theresulting litters screened for under-glycosylated cells having theconstruct. By providing for a different phenotype of the blastocyst andthe ES cells, chimeric progeny can be readily detected.

[0117] The chimeric animals are screened for the presence of themodified gene and males and females having the modification are mated toproduce homozygous progeny. If the gene alterations cause lethality atsome point in development, tissues or organs can be maintained asallergenic or congenic grafts or transplants, or in in vitro culture.

Transgenic Animals with Inducible Endogenous Sequences

[0118] Transgenic animals with an inducible endogenous genes that may bedetrimental at some point in development may be manipulated for purposesof the invention by placing the under-glycosylated PrP under aninducible promoter. For example of PrP expression using such systems,see U.S. Ser. No. 09/052,963 which is incorporated herein by reference.Such animals would not express the under-glycosylated PrP gene until itwas necessary for purposes of the assay, thus allowing the animals tosuppress any potential effects of the transgene when such expression isunnecessary. For example, the expression of the under-glycosylated PrPallele can be suppressed throughout development, and then expression ofthe PrP gene can be used to advance progression of the disease in matureanimals. The establishment of a transgenic line free from potentialdevelopmental problems but with the ability to rapidly form prions inresponse to a contaminated sample would be a major advantage in assaysto detect prions in a sample.

[0119] The creation of transgenic animals with altered endogenous PrPgene is described in U.S. Pat. No. 5,698,763 to Weissmann. It has beenproposed to disrupt the endogenous PrP gene completely in animals toprevent development and progression of prion-mediated disease. Thetransgene encoding the PrP of the present invention may be introducedinto animals with a PrP^(0/0) background, thus leaving theunder-glycosylated PrP as the only PrP expressed in the transgenicanimal. Although the under-glycosylated PrP gene will allow infection bydifferent strains of prions, and thus a species-specific transgene isnot a requirement, the under-glycosylated PrP can be tailored dependingon the species that is to be tested. For example in human testing anunder-glycosylated human PrP transgene can be used and for bovinetesting an under-glycosylated bovine transgene can be used, and inparticular specific polymorphisms and/or mutations can be introduced tostudy particular prion-mediated disorders, e.g. mutations found in humanCJD can be altered in the transgene.

[0120] The under-glycosylated PrP transgene of the present inventionwill allow the study of progression of prion-mediated disorders in ananimal model which has a much shorter period of incubation for suchdiseases. Such studies may examine biological phenomena such as thelevels of expression needed for physiological symptoms to appear, thereversibility of the morphological symptoms following initial stages ofthe disease, and the like.

Test Animal

[0121] Although a variety of different test animals could be used fortesting for the presence of prions within a sample, preferred hostanimals are mice and hamsters, with mice being most preferred in thatthere exists considerable knowledge on the production of transgenicanimals.

[0122] Other possible host animals include those belonging to a genusselected from Mus (e.g. mice), Rattus (e.g. rats), Oryctolagus (e.g.rabbits), and Mesocricetus (e.g. hamsters) and Cavia (e.g., guineapigs). In general mammals with a normal full grown adult body weight ofless than 1 kg which are easy to breed and maintain can be used. Thehost PrP gene can be changed to include codons from genetically diversePrP genes from test animals belonging to a genus selected from Bos,Ovis, Sus and Homo. Preferably, a mouse host PrP gene is changed toinclude codons from a human, cow or sheep PrP gene, with human beingmost preferred. Humans are preferred because an important object of theinvention is to use the animal to test a sample of material to determineif that material has prions which will infect a human and cause a humanto develop a CNS disease such as CJD. Preferred transgenic animals aredisclosed in U.S. Pat. No. 5,565,186 issued Oct. 15, 1996 and WO97/04814 published Feb. 13, 1997 which are incorporated herein byreference to disclose transgenic animals and methods of making and usingsuch.

[0123] The genetic material which makes up the PrP gene is known for anumber of different species of animals (see Gabriel et al., Proc. Natl.Acad. Sci USA 89:9097-9101 (1992)). Further, there is considerablehomology between the PrP genes in different mammals. For example, seethe amino acid sequence of mouse PrP compared to human, cow and sheepPrP in FIGS. 3 +L, 4 and 5 wherein only the differences are shown.Although there is considerable genetic homology with respect to PrPgenes, the differences are significant in some instances. Morespecifically, due to small differences in the protein encoded by the PrPgene of different mammals, a prion which will infect one mammal (e.g. ahuman) will not normally infect a different mammal (e.g. a mouse). Dueto this “species barrier,” it is not generally possible to use normalanimals, (i.e., animal which have not had their genetic material relatedto prions manipulated) such as mice to determine whether a particularsample contains prions which would normally infect a different speciesof animal such as a human.

Endogenous PrP Status of Test Animals

[0124] Commercially useful transgenic animals are preferably small andeasy to reproduce; thus, host animals such as mice, hamsters, guineapigs and rats are preferred, with mice being most preferred. In orderfor the transgenic animals to be useful, it is necessary for the animalsto be susceptible to infection with prions which normally infect onlygenetically diverse test animals, and in particular animals ofcommercial significance for testing, such as humans, cows, deer, horses,sheep, pigs, cats, dogs and chickens, with humans being most preferred.Further, for the transgenic and hybrid animals to be useful in apractical and commercial sense, it is necessary for the animals todemonstrate symptoms of the disease within a relatively short periodafter inoculation, and for a very high percentage of the animals todemonstrate symptoms of the disease after inoculation.

[0125] When the entire PrP gene of a test animal (such as a human) ismade functional in the host animal (such as a mouse) the resultingtransgenic animal (with a low copy number of human PrP genes) is notsusceptible to infection with human prions. Infection would occur if theendogenous PrP gene of the host animal is ablated. Furthermore, if onlysome of the codons differing between the host and the test animal areswitched, the resulting transgenic animal is susceptible to infectionwith prions which normally only infect the test animal.

[0126] As the copy number of the artificial gene in the transgenicanimal is increased, the incubation time in some cases decreases. Inaddition, certain genetic defects resulting in prion diseases havedifferent genetic defects in their PrP gene, and by matching the defectsin any transgenic animal will render that animal more susceptible toinfection with prions from the diseased human.

[0127] With this knowledge, we deduced that it is possible to produce atransgenic animal wherein all of the PrP gene of the host animal isreplaced with the PrP gene of a test animal with an attached induciblesequence, thus obtaining a useful transgenic animal which is susceptibleto infection with prions which normally only infect the test animal bysubstantially increasing the copy number of the test animal's PrP genein the host animal and preferably also ablating the endogenous PrP gene.The copy number can be substantially increased without affecting thesurvivability of the developing animal by turning expression off duringdevelopment.

[0128] Based on the above, it can be understood that the preferredstatus of the endogenous gene of the transgenic animals are (1) animalssuch as mice which include a chimeric PrP gene, i.e., only a portion,but not all, of their PrP gene replaced with a corresponding portion ofthe under-glycosylated glycosylation sites or (2) animals with anablated endogenous PrP gene and a PrP gene from another animal such as ahuman most preferable where that human PrP gene has a mutation whichprevents proper glycosylation of the molecule. These genotypes enhancethe ability of the inducible transgenic system to detect prioninfectivity of a genetically diverse animal with a shorter incubationperiod. Details regarding construction and testing of animals witheither chimeric PrP genes or ablated endogenous PrP genes can be foundin U.S. Ser. No. 08/509, 261, which is incorporated herein in itsentirety.

In vivo Drug Efficacy Evaluation

[0129] Transgenic animals of the invention can be used to evaluate theefficacy of drugs. In its simplest form, the exogenous gene is expressedin two mice and the drug is administered to one but not the other mouse.The effect of the drug on inhibiting the progress of disease as comparedto the mouse with no drug is then determined. Measurements are takenover time for both mice to determine the rate at which harmful proteinis cleared from the system of each mouse. The drug is determinedefficacious if it increases the rate at which the harmful protein iscleared from the system as compared to the rate it is cleared from thesystem of the mouse with no drug.

Evaluation of Locomotor and Motor Coordination Deficits for RapidDiagnosis of Cerebellar Scrapie Progression

[0130] The diagnosis of scrapie in rodents involves the detection of atleast two classical neurological signs associated with prion diseases aswell as the progression of these signs over time. Classical clinicalsigns are: agressivity, ataxia, dysmetria, tremor, head-bobbing, lack ofrighting reflex, convulsions, kyphosis, head tilt, tail rigidity,bradykinesia, proprioceptive deficits, masked facies, loss of deep painsensation, circling and paralysis. The use of a system of the presentinvention, whether inducible or not, where PrP expression ispredominantly localized within the cerebellum, facilitates the detectionof replicating prions as the neurodegeneration can be followed byscoring ataxia or disorders of movement, gait, equilibrium andcoordination associated with cerebellar degeneration.

[0131] To diagnose animals exhibiting scrapie sickness in a shorterperiod of time, a pressure-sensitive measurement system may be employedto detect and record an inoculated animal's physiological changes, andspecifically its motor skills. One example of such a pressure sensitivesystem that would be effective in monitoring changes in walking is aplatform with a piezoelectric ground reaction force (GRF) measurementsystem such as that used in the Gaitway Instrumented Treadmill (KistlerBiomechanics, Winterthut, Switzerland). Such a system has the ability tomeasure vertical ground reaction force and center of pressure forcomplete, multiple foot strikes. The system also has an integratedsoftware system that can distinguish between left and right strikes andmeasure vertical force, center of pressure, and temporal gaitparameters. A database can keep track of trials by subject name,identification number, or other user-specified classification. Multipletrials can be overlaid on a single graph, and the progression of asingle subject examined over time. The time base can be varied to viewdata in absolute time, relative time, percent contact, percent step, andpercent gait cycle.

[0132] Using such systems for the early detection of scrapie disease canalso be achieved more by the systematic quantitative evaluation of theseparameters over time using apparatus designed for such purpose.

[0133] Another method of evaluating motor coordination is a rotorod test(Sakaguchi et al.). Animals are placed on a rotating rod, whose rotatingfrequency is steadily increased from 10 rpm until the animals falls fromit. The animal is subjected to ten trials and its best score recorded asits score on that given day. This test would offer a quantitativemeasurement of motor skills over the progression of prion disease andpermit the early detection of motoric deficiencies. Other systems withthe ability to distinguish motor skill abilities may also be employed,as will be obvious to those skilled in the art.

Standardized Prion Preparation

[0134] For both cellular and animal assays used to study prionprogression, infectivity, and/or the mechanistic interactions involvedin prion disease, standardized preparations of prions may be used todecrease the variability of the studies. Although the preparation can beobtained from any animal it is preferably obtained from a host animalwhich has brain material containing prions of a test animal. Forexample, a Tg mouse containing a human prion protein gene can producehuman prions and the brain of such a mouse can be used to create astandardized human prion preparation. The preparation can be furtherstandardized, by repeating the above process. More specifically, per theabove process some prion containing material must be used to inoculatethe transgenic mice. The source of that prion containing material mayitself be unpredictable and result in infecting transgenic mice indifferent ways. Thus, if the transgenic mice are infected with anonstandard material some may develop the symptoms of prion disease atdifferent rates and some may not develop symptoms at all. If a group ofmice which develops symptoms of prion disease at the same time aresacrificed and their brains extracted and homogenized such will create arelatively standard prion preparation.

[0135] In that the preparation is to be a “standard” it is preferablyobtained from a battery (e.g., 100; 1,000, or more animals) ofsubstantial identical animals. For example, 100 mice all containing avery high copy number of human PrP genes (all polymorphisms andmutations) would spontaneously develop disease and the brain tissue fromeach could be combined to make a useful standardized prion preparation.The preparation of such standards, and animals useful in the productionof such standards, is disclosed in U.S. Ser. No. 09/199,523, which isincorporated herein by reference.

[0136] By using standardized prion preparations it is possible to createextremely dilute compositions containing the prions. For example, acomposition containing one part per million or less or even one part perbillion or less can be created. Such a composition can be used to testthe sensitivity of the transgenic mice of the invention in detecting thepresence of prions in the sample.

[0137] Prion preparations are desirable in that they will include aconstant amount of prions and are extracted from an isogeneicbackground. Accordingly, contaminates in the preparations will beconstant and controllable. Standardized prion preparations will also beuseful as controls in the carrying out of bioassays in order todetermine the presence, if any, of prions in various pharmaceuticals,whole blood, blood fractions, foods, cosmetics, organs and in particularany material which is derived from an animal (living or dead) such asorgans, blood and products thereof derived from living or dead humans.Thus, standardized prion preparations will be valuable in validatingpurification protocols where preparations are spiked and reductions intiter measured for a particular process.

EXAMPLES

[0138] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to make and use the present invention, and are not intended to limitthe scope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

EXAMPLE 1

[0139] Mutations are introduced into human PrP sequences to alter twoknown glycosylation sites of the molecule. A mouse-hamster chimeric PrPtransgene (MHM2) is mutagenized to contain a double mutation where thetwo glycosylation sites are substituted from asparagine to glutamine(N180Q/N196Q), and cloned into a pSPOX expression vector. The PrPmolecule was mutagenized using a mismatched oligonucleotide primer andPCR amplification. Following amplification, the mutated PrP DNA isdigested with unique restriction endonucleases to allow directionalcloning into the pSPOX vector. These expression vectors were thentransiently expressed in neuroblastoma cells (N2a cells) andsubsequently infected with 10% brain homogenates from experimentallyscrapie-infected mice.

[0140] After 4 days of incubation, appearance of newly formed prions canbe seen after cell lysis, proteinase-digestion and immunoblot. The blotwas developed using 3F4 (which sepcifically detects the MHM2 construct)as the primary antibody, and detection was carried out using the ECLsystem. De novo formed prions can be distinguished from residual prionsof the inoculate by an epitope-tagging of the under-glycosylated,transfected PrP.

[0141] The proteinase K-resistant fragment of PrP^(Sc) was found incultures expressing the glycosylation mutant and exposed to homogenatesfrom mice infected with three different prion strains: RML, ME7 and301V(B). Conversion of unglycosylated PrP was blocked by the Q218Kmutation. The results showed that all strains of mouse prions tested canconvert unglycosylated PrP into prions. No protease-resistant PrP wasseen when cells were incubated with no brain homogenates or those fromnormal mice (CD-1).

[0142] When cells were transiently transfected with pSPOX MHM2-PrP(N180Q, N196Q; Q218K), and incubated with a 10% RML mouse-scrapie brainhomogenate, no conversion to protease-resistance was seen; thisadditional mutation at the C-terminus is thought to block an essentialconversion co-factor, protein X. The effect of the additional Q218Kmutation demonstrates that conversion of under-glycosylated PrP isperformed as for wild-type PrP with the help of co-factors, and not ametastability of conformation or a protease-resistance caused by bindingof inoculum-derived PrP^(Sc) to under-glycosylated PrP.

[0143] When cells were transiently transfected with pSPOX MHM2-PrPwild-type and incubated with a 10% RML mouse-scrapie brain homogenate,almost no protease-resistant PrP could be seen. One very faint bandseems to show up at the height of the under-glycosylated PrP,demonstrating that from the different glycoforms of wild-type PrP, it isalso the under-glycosylated form that is (although far less efficiently)converted.

[0144] These experiments show that prions can be rapidly replicated andamplified in a cell culture system and used to amplify trace amounts ofprions in suspected tissues.

[0145] While the present invention has been described with reference tothe specific embodiments thereof, it should be understood by thoseskilled in the art that various changes may be made and equivalents maybe substituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1. An isolated PrP protein characterized by: an amino acid sequencedifferent from wild-type PrP; a reduction in glycosylation relative towild-type PrP; and cellular localization sufficient to allow prioninfection.
 2. The PrP protein of claim 1, wherein the amino acidsequence is different at one or more positions.
 3. The PrP protein ofclaim 1, wherein an asparagine in at least one glycosylation site issubstituted with a different amino acid.
 4. The PrP protein of claim 3,wherein the asparagine is substituted by a glutamine.
 5. The polypeptideof claim 4, comprising an amino acid sequence of SEQ ID NO:2.
 6. Anisolated nucleic acid sequence or complement thereof encoding a PrPprotein characterized by a reduction in glycosylation relative towild-type PrP and cellular localization sufficient to allow prioninfection.
 7. The isolated nucleic acid of claim 6 having the sequenceof SEQ ID NO:
 1. 8. A recombinant host cell having operatively insertedtherein an exogenous nucleic acid sequence encoding the protein ofclaim
 1. 9. The recombinant host cell of claim 8, wherein the nucleicacid is stably inserted into the genome of said cell.
 10. An assay,comprising the steps of: contacting a sample putatively comprisingprions with a recombinant cell line, wherein the cell line hasoperatively inserted therein an exogenous nucleic acid sequence encodinga protein characterized by an amino acid sequence different fromwild-type PrP, a reduction in glycosylation relative to wild type PrP,and cellular localization sufficient to allow prion infection; anddetermining the presence of prions in the sample after contacting thecell line.
 11. The assay of claim 10 wherein the determining is carriedout with an antibody.
 12. The assay of claim 10 wherein the determiningis carried out with a transgenic mouse.
 13. A transgenic animalcharacterized by a genome having operatively inserted therein anexogenous PrP transgene comprising the nucleic acid of claim
 6. 14. Thetransgenic animal of claim 13, further comprising a genome wherein atleast one allele of an endogenous PrP gene is altered.
 15. Thetransgenic animal of claim 13, wherein both alleles of the endogenousPrP gene are ablated.
 16. A non-human transgenic animal, comprising: agenome artificially altered to comprise an exogenous PrP nucleic acid ofclaim 5 and an inducer sequence which affects expression of theexogenous PrP gene.
 17. The transgenic animal of claim 16, wherein theanimal is selected from the group consisting of a mouse, a rat and ahamster and the genetically diverse animal is selected from the groupconsisting of a human, cow, sheep, horse, pig, chicken, dog or cat. 18.The transgenic animal of claim 16, wherein the inducer system comprisesan inducible transactivator sequence and an operator sequence whereinboth are operably linked to the exogenous PrP gene in a manner so as tocontrol expression of the exogenous PrP gene.
 19. A method for detectingprion infectivity of a sample, the method comprising the steps of:obtaining sample from an animal to be tested; inoculating the transgenicanimal of claim 13 with the sample; and observing the transgenic animalfor symptoms of prion disease.
 20. The method of claim 19, wherein thetest animal is selected from the group consisting of human, cow, sheep,pig, horse, cat, dog, turkey or chicken, and the transgenic animal isselected from the group consisting of: mice, rats, rabbits, hamsters andguinea pigs.
 21. A method for identifying a biologically active agentthat modulates infection or progression of a prion-mediated disorder,the method comprising: combining a candidate agent with a detectionsystem selected from the group consisting of: (a) a mammalian PrPpolypeptide characterized by a reduction in glycosylation relative tothe wild-type PrP polypeptide and proper cellular localization; (b) acell comprising a nucleic acid encoding a mammalian PrP polypeptidecharacterized by a reduction in glycosylation relative to the wild-typePrP polypeptide and proper cellular localization; (c) a non-humantransgenic animal model having a genome comprising a mammalian PrPpolypeptide characterized by a reduction in glycosylation relative tothe wild-type PrP polypeptide and proper cellular localization;inoculating the detection system with a prion preparation; anddetermining the amount of protease resistant prions; wherein the levelof protease-resistant prions is indicative of the effect of thecandidate agent on prion-mediated disorders.
 22. A process fordiagnosing a patient suspected of having a prion-mediated disordercomprising the steps of: a) obtaining cells or body fluids from thepatient; b) transfecting said cells with a PrP nucleic acid of claim 3;and c) detecting prions in the cells.